This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We intend to use techniques that are more efficiently time-resolved to study the dynamics of protein:protein interactions. Additionally, we want to use advanced imaging techniques to detail where in the cell changes in FRET are occurring. Our studies will continue to focus primarily on the IFN-gamma receptor complex, with extensions into the IL-10 and interferon-gamma (IFN-g) receptor complexes. The IFN-g receptor complex is especially important as its crosstalk and biological synergy with the IFN-g receptor complex has been suggested. IFN-g is representative of a group of proteins called Type I IFNs that are released by cells when infected by virus, and function to protect nonhematopoietic cells from virus infection. The specific aims are: 1. To investigate the dynamics of receptor chain interactions in live cells. 2. To investigate the dynamics of interactions of Jak kinases with receptor chains in live cells. 3. To investigate the kinetics of receptor movements, Jak kinase movements and Stat movements. 4. To analyze the interactions among receptor complexes when bound by ligand. Previously we intended to use Fluorescence Resonance Energy Transfer (FRET) to ascertain the gross structure of the interferon-gamma (IFN-g) receptor complex in its native environment in live cells. We employed this direct approach because conflicting conclusions were obtained from studies of the receptor complex outside its natural environment. The system setup at the Regional Laser Biotechnology Laboratory is ideally suited for FRET microscopy: a confocal microscope is coupled to a monochromator, allowing frequency-resolved fluorescence measurements to be done on biological samples without any additional manipulations. Frequency-resolved measurements are essential in the unequivocal establishment of FRET in fluorescence emission. The elucidation of the kinetics of the IFN-g signal transduction cascade as it exists in real cells will be of great importance in the characterization of inhibitors or activators of IFN-g signaling that could have therapeutic use in modulating the immune system in cancer, autoimmune disease, viral disease, tissue transplant rejection, and other cases. We followed the kinetics of Stat1 activation by the IFN-g receptor complex and found that the kinetics were very unusual: under conditions where ligand-binding occurs almost instantaneously, a latency period in the activation of Stat1 exists unless either the IFN-gR1 chain or the IFN-gR2 chain is in excess. One explanation of this latency period that is supported by previously published observations is that two ligand-bound IFN-g receptor complexes are interacting. The use of real-time fluorescent techniques will be valuable in establishing the kinetic process of receptor activation and movement.